Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

103

39-POS

Board 39

On the Hyperpolarization Activated Ion Current of Xenpus Oocytes, Its Relation to HCNs

Expression and Gating Properties of HCN2 Mutants

Ricardo Hernández

, Adriana Pétriz, Edith A. Espino-Saldaña, Adán Hernández, Ataúlfo

Martínez-Torres.

Instituto de Neurobiologia, UNAM, Juriquilla, Querétaro, Mexico.

Frog oocytes present a diversity of ion conductances including one that is activated upon extreme

negative hyperpolarizating potentials. In an attempt to identify the molecular component of this

current, the four cDNAs that code for the Xenopus tropicalis hyperpolarization activated cyclic

nucleotide-gated channels (HCN1-4) were cloned, expressed in HEK293 cells and single-site

mutants of a conserved series of amino acids neighboring the selectivity filter were generated

and studied

The ion currents induced by hyperpolarizing pulses were recorded using two-electrode, voltage-

clamp in oocytes of X. tropicalis, stepping the membrane from -60 mV to -140. The genome

sequence of this frog was explored to determine the presence of HCN-encoding genes, cDNAs

were identified, cloned, introduced into expression plasmids, and transfected in HEK293 cells or

used as templates to produce mRNA for expression in X. laevis oocytes and recorded by voltage-

clamp

In X. tropicalis oocytes the current activates and inactivates slowly. The current-voltage

relationship showed an inward rectification at potentials more negative than -130 mV and the

reversal potential was -23 +/- 1.5 mV. Antisense oligonucleotides to HCN1-4 did not block the

ion current. The cDNAs of HCN1, 2 and 3 resulted as predicted from GeneBank whereas HCN3

forms a truncated channel predicted to be non-functional and was named HCN-like. HCN-like,

co-expressed with the other HCNs, blocked the generation of functional channels. Finally, the

HCN2 mutant LCI -> AAA shows changes in the activation and inactivation kinetics, in

activation threshold and potassium modulation

In conclusion, we show that HCN RNAs are expressed in X. tropicalis oocytes. The HCN-like

RNA heteromerizes with other HCNs and blocks their expression. The 391LCI393 amino acid

sequence of HCNs is important to provide an effective gating of the ion channel